PROJECT SUMMARY/ABSTRACT Vertical transmission of cytomegalovirus (CMV) is the single most common cause of congenital infection worldwide, often resulting in deafness and neurodevelopmental delay for afflicted children. CMV related neurologic complications are more frequent and severe following primary maternal infection during pregnancy, and therefore a vaccine to prevent maternal acquisition of CMV during pregnancy is a potential strategy to reduce the incidence of infant congenital disease. The most successful maternal immunization regimen tested to date is a subunit vaccine consisting of CMV glycoprotein B (gB) combined with MF59 adjuvant, which achieved a promising 50% efficacy in multiple phase II clinical trials. However efforts to improve this vaccine have been slowed by an incomplete understanding of the determinants of protection against CMV infection. Previous studies have revealed a direct correlation between magnitude of the maternal CMV neutralizing antibody response and risk of congenital CMV infection. Concordant with these earlier studies, our laboratory has generated compelling data using both clinical cohorts and a novel nonhuman primate model of congenital CMV transmission, demonstrating that antibodies with the ability to neutralize CMV viruses may protect against in utero transmission. Yet it remains unclear whether antibody responses targeting the gB protein alone, and not other CMV glycoproteins, are sufficient for an effective vaccine to prevent congenital CMV. Recent discovery of the CMV gB crystal structure and identification of distinct protein domains targeted by neutralizing antibodies have made it possible to investigate gB/MF59-elicited protective antibody responses. Importantly, CMV surface glycoproteins such as gB have a high degree of structural diversity, suggesting that mutation of these proteins may be a mechanism to evade vaccine-elicited immune responses. I hypothesize that vaccine efficacy of a maternal gB immunization strategy is dependent upon 1) neutralizing antibodies targeting gB domains and 2) viral immune evasion from these protective immune responses. Over the next two years as a component of my Ph.D. dissertation project, I propose to employ novel approaches and cutting- edge vaccinology techniques to assess both antibody and viral determinants of vaccine protection using two independent gB/MF59 phase II trial cohorts. First, I will assess whether neutralizing antibodies predict CMV acquisition risk and define the epitope specificities of those protective antibodies. Secondly, through deep sequencing of viral variants and subsequent viral ?sieve analysis,? I will investigate sequence diversity at the loci of gB domains to determine if viral immune evasion of vaccine-elicited immune responses is associated with vaccine failure. Finally, I will engineer and characterize chimeric rhesus CMV viruses containing human gB neutralizing domains for validation of viral immune evasion at identified polymorphic sites. These chimeric viruses will be used in the future in a nonhuman primate model developed by our laboratory, setting the stage for rational design and testing of a glycoprotein-based vaccine for the elimination of congenital CMV infection.